Amino Acid‐Based Advanced Liquid Formulation Development for Highly Concentrated Therapeutic Antibodies Balances Physical and Chemical Stability and Low Viscosity

Abstract

To develop highly concentrated therapeutic antibodies enabling convenient subcutaneous application, well stabilizing pharmaceutical formulations with low viscosities are considered to be key. The purpose of this study is to select specific amino acid combinations that reduce and balance aggregation, fragmentation and chemical degradation, and also lower viscosity of highly concentrated liquid antibodies. As a model, the therapeutically well-established antibody trastuzumab (25->200 mg mL ) in liquid formulation is used. Pre-testing of formulations based on a stabilizing and protecting solutions (SPS®) platform is conducted in a thermal unfolding model using differential scanning fluorimetry (DSF) and accelerated aging at 37 and 45 °C. Pre-selected amino acid combinations are further iteratively adjusted to obtain stable highly concentrated antibody formulations with low viscosity. Size exclusion chromatography (SE-HPLC) reveals significantly lower aggregation and fragmentation at specific amino acid:sugar and protein:excipient ratios. Dynamic viscosities <20 mPa * s of highly concentrated trastuzumab (≥200 mg mL ) are measured by falling ball viscosimetry. Moreover, less chemical degradation is found by cationic exchange chromatography (CEX-HPLC) even after 6 months liquid storage at 25 °C. In conclusion, specifically tailored and advanced amino acid-based liquid formulations avoid aggregation and enable the development of stable and low viscous highly concentrated biopharmaceuticals.

Aggregation of protein therapeutics enhances their immunogenicity: causes and mitigation strategies

“…To control protein stability, both during production and in the final formulation of the therapeutic, environmental factors such as pH, ionic strength, temperature and mechanical agitation need to be considered, as well as the addition of excipients in the formulation. 89 Excipients are all the components of a drug formulation other than the active drug itself, including salts and buffer system to control pH and osmolality, as well as other added compounds that for instance reduce viscosity, 90 act as preservatives 88 or that enhance protein stability through a variety of mechanisms. 91,92 Controlling sources of stress Environmental conditions during production, processing, transportation and storage of a therapeutic protein can enhance aggregation by affecting either (or both) conformational and colloidal stability.…”

“…Apart from controlling environmental sources of protein stress, there are numerous excipients that hinder protein denaturation, for instance disaccharides, such as sucrose 62 and trehalose. 90 In solution these are osmolytes and act as stabilizers by preferential exclusion; [108][109][110] i.e. by creating a highly polar environment surrounding proteins, thus inhibiting the exposure of hydrophobic pockets hidden by the native fold.…”

“…35,133 Moreover, excipients can cause oxidation, for instance polyoxyethylene-based surfactants (PS20 and PS80) contain ether linkages and unsaturated alkyl chains that can auto-oxidize to form reactive alkyl peroxides, which in turn cause oxidative changes to the protein. 140,141 To prevent oxidative protein changes, the use of chelators 92,142,143 to prevent metal-induced oxidation or antioxidants 40,90,137,144,145 have been demonstrated to impede protein oxidation and aggregation.…”

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

“…For example, polysorbates are commonly used in biologics as a stabilizing agent; however, their addition in high concentrations can denature proteins and cause adverse side effects such as injection site reactions [114,115,116]. Injectable mAb formulations are co-formulated with recombinant human hyaluronidase, specifically as a permeation enhancer for more efficient absorption into tissue, although the inclusion of this additional biologic adds further burden to the formulation’s viscosity and propensity to aggregate [5,8,25,26,27,28,117,118,119,120,121,122,123]. Antibody therapies for IV administration are prepared as lyophilised powder for reconstitution and further dilution, and injectable administrations are prepared as liquid-based formulations in pre-filled syringes.…”

Algorithm-Based Liquid Formulation Development Including a DoE Concept Predicts Long-Term Viral Vector Stability

“…We recently demonstrated that specifically tailored amino acidebased formulations have a high potential to avoid stressmediated degradation of complex molecules such as monoclonal antibodies [22][23][24] and vaccines. 25 This formulation approach has been shown in the past to stabilize a broad spectrum of target molecules during drying and reconstitution as well as during liquid storage, according to the concepts of preferential binding and preferential exclusion, [26][27][28][29] respectively.…”